Bis-(hydroxylakylamino)-anthraquinone inhibitors of protein kinase C

ABSTRACT

##STR1## The present invention provides novel substituted anthraquinones having formula (α), wherein R 1  and R 2  are independently H, C 1  -C 10  alkyl, aryl, arylalkyl, alkylaryl, or R 1  and R 2  taken together with the adjacent nitrogen atom N form a substituted or unsubstituted cyclic ring which may contain additional heteroatoms; n and m are independently 1, 2, or 3; A is halogen, OH, alkoxy, OCO(NR 3  R 4 ), S--C(NH 2 )=NR 5 , or when m=1, comprises an oxirane ring with the adjacent oxygen atom; R 3 , R 4 , and R 5  are independently H, alkyl, or aryl; X is H, OH, NR 6  R 7 , Cl, Br, I, F, alkyl, aryl, alkoxy, aryloxy, COOR 8 , or CONR 9  R 10  ; and R 6 , R 7 , R 8 , R 9 , and R 10  are independently H, lower alkyl, or aryl useful for inhibiting protein kinase C and treating conditions related to, or affected by inhibition of protein kinase C, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S.application Ser. No. 07/609,252, filed Nov. 5, 1990 in the names of JackB. Biang and Mary George Johnson entitled"BIS-(HYDROXYALKYLAMINO)-ANTHRAQUINONE INHIBITORS OF PROTEIN KINASE C",which is in turn a continuation in part application of U.S. applicationSer. No. 07/609,179, filed Nov. 2, 1990 now abandoned, the disclosuresof both of which are hereby incorporated by reference as if fully setforth herein.

FIELD OF THE INVENTION

The present invention relates to the field of treatments forinflammatory and cardiovascular diseases, and chemotherapeutic agents.More particularly, the present invention relates to novel asymmetric1,4-bis-(amino-hydroxylalkylamino)-anthraquinones for inhibiting theenzyme protein kinase C.

BACKGROUND OF THE INVENTION

Protein kinase C (PKC) is a family of calcium stimulatable andphospholipid-dependent serine/threonine-specific protein kinases whichplay an important role in cellular growth control, regulation, anddifferentiation. protein kinase C is also fundamental to the processesinvolved in tumorigenicity, since it is the major high-affinity receptorfor several classes tumor promoters as well as for endogenous cellulardiacylglycerols. These tumor promoters also stimulate protein kinase Ccatalysis. Castagna et al. (1982) J. Biol. Chem. 257: 7847 reporteddirect activation of protein kinase C by tumor-promoting phorbol esters.The mechanisms of protein kinase C action have been described in U.S.Pat. No. 4,816,450 issued Mar. 28, 1989 to Bell et al., the disclosuresof which are specifically incorporated as if fully set forth herein.protein kinase C is activated by diacylglycerol (DAG), a neutral lipid,and when activated will transfer the γ-phosphate of MgATP to a serine orthreonine residue on a substrate protein.

Since the activation of protein kinase C have been implicated in severalhuman disease processes, including cancer tumors, inflammation, andreperfusion injury, inhibition of protein kinase C should be of greattherapeutic value in treating these conditions.

Protein kinase C inhibitors have been reported to potentiate theantitumor activity of cis-platin both in vitro and in vivo (Grunicke etal. (1989) Adv. Enzyme Regul. 28: 201; and German Offenlegungsschrift DE3827974). In addition, it has been suggested that protein kinase C wouldbe a potential target for therapeutic design because of its central rolein cell growth (Tritton, T. R. and Hickman, J. A. Cancer Cells 2: 95-102(1990)).

Protein kinase C inhibitors have been demonstrated to block plateletaggregation and release of neutrophil activating agents such as plateletactivating factor (PAF) (Schachtele et al. (1988) Biochem. Biophy. Res.Commun. 151: 542; Hannun et al. (1987) J. Biol. Chem. 262: 13620; Yamadaet al. (1988) Biochem. Pharmacol. 37: 1161). protein kinase C inhibitorshave also been shown to inhibit neutrophil activation, and chemotacticmigration (McIntyre et al. (1987) J. Biol Chem. 262: 15730; Lambreth etal. (1988) J. Biol. Chem. 263: 3818; Pittet et al. (1987) J. Biol. Chem.262: 10072; and Gaudry et al. (1988) Immunology 63: 715), as well asneutrophil degranulation and release of proteolytic enzymes and reactiveoxygen intermediates (Wilson et al. (1986) J. Biol. Chem. 261: 12616;Fujita et al. (1986) Biochem. Pharmacol. 35: 4555; Berkow et al. (1987)J. Leukoc., Biol. 41: 441; Salzer et al. (1987) Biochem. Biophys. Res.Commun. 148: 747; Kramer et al. (1989) J. Biol. Chem. 262: 5876; andDewald et al. (1989) Biochem. J. 264: 879). Thus inhibitors of proteinkinase C have the capability of blocking all three of the mostsignificant mechanisms of pathogenesis associated with myocardialreperfusion injury, and should thus have a decided therapeuticadvantage. Additionally, the inhibitory effect of protein kinase Cinhibitors on keratinocytes, and on the oxidative burst in neutrophilswill lead to an anti-inflammatory effect.

Substituted anthraquinones have been reported for various uses,including cancer treatment. U.S. Pat. No. 3,960,751 issued Jun. 1, 1976to Moriyama et al. discloses substituted anthraquinones useful aspleochroic dyes. U.S. Pat. No. 4,598,155 issued Jul. 1, 1986 to Adamdiscloses tetrazole substituted anthraquinones useful as dyes.

U.S. Pat. No. 4,762,648 issued Aug. 9, 1988 to Stache et al. disclosesmono-functional and bis-functional anthraquinone-(oxy-2,3-oxidopropanes)useful as intermediates in the preparation of drugs possessingβ-receptor blocker action and as crosslinking agents in the preparationof polymers. The compounds also exhibit cytostatic activity.

Several patents disclose the use of substituted anthraquinones fortreatment of neoplasms. U.S. Pat. No. 4,894,451 issued Jan. 16, 1990 toKrapcho et al. discloses unsymmetrical1,4-bis-(aminoalkylamino)-anthracene-9,10-diones useful in the treatmentof neoplasms. U.S. Pat. No. 4,310,666 issued Jan. 12, 1982 to Zee-Chenget al discloses 1,4-bis-(substituted aminoalkylamino)-anthraquinonesuseful in the treatment of neoplasms. U.S. Pat. No. 4,526,989 issuedJul. 2, 1985 to Murdock et al discloses symmetrical1,4-bis(substituted-amino)-5,8-dihydroxyanthraquinones useful aschelating agents and for inhibiting the growth of tumors. U.S. Pat. No.4,197,249 issued Apr. 8, 1980 to Murdock et al also disclosessymmetrical 1,4-bis(substituted-amino)-5,8-dihydroxyanthraquinonesuseful as chelating agents and for inhibiting the growth of tumors. U.S.Pat. No. 4,540,788 issued Sep. 10, 1985 to Murdock discloses1,4-bis[-aminoalkyl)amino]-9,10-anthracenediones and leuco bases thereofwhich are useful as chelating agents and for inducing regression ofleukemia and/or inhibition of tumor growth in mammals. Japanese patent19819 issued May 7, 1990 discloses substituted anthraquinones havingsubstituents at positions 5 and 6 of the anthraquinone ring structurewhich are useful as anti-tumor agents alone or in combination with otheranti-tumor agents.

German Offenlegungsschrift DE 3827974 A1 discloses therapeuticpreparations comprising a protein kinase C inhibitor in combination witha lipid, a lipid analogue, a cytostatic agent or phospholipase inhibitoruseful for cancer therapy. However, none of the protein kinase cinhibitors disclosed in this publication are substituted anthraquinones.

Although substituted anthraquinones have been reported for cancertreatments, substituted anthraquinones such as Mitoxantrone, are knownto be associated with side effects mainly immunosuppressive activity,and drug resistance. Accordingly novel cancer treatments that avoid someor all of these drawbacks are needed.

Further, inflammation and reperfusion injury, particularly pertaining tocardiac injury, are common conditions for which there exists nodefinitive treatment despite extensive research and appropriatetreatments for these conditions are needed.

SUMMARY OF THE INVENTION

The present invention provides novel asymmetric substitutedanthraquinones having the formula ##STR2## wherein R₁ and R₂ areindependently H, C₁ -C₁₀ alkyl, aryl, arylalkyl, alkylaryl, or R₁ and R₂taken together with the adjacent nitrogen atom N form a substituted orunsubstituted cyclic ring, which ring may optionally contain additionalheteroatoms including oxygen, nitrogen or sulphur; n and m areindependently 1, 2, or 3; A is Halogen, OH, alkoxy, OCO(NR₃ R₄),S--C(NH₂)═NR₅, or when m=1, comprises an oxirane ring with the adjacentoxygen atom; R₃, R₄, and R₅ are independently H, alkyl, or aryl; X is H,OH NR₆ R₇, Cl, Br, I, F, alkyl, aryl, alkoxy, aryloxy, COOR₈, or CONR₉R₁₀ ; and R₆, R₇, R₈, R₉, and R₁₀ are independently H, lower alkyl, oraryl.

The novel compounds of the invention are useful for inhibiting proteinkinase c and are further useful for treating conditions related to, oraffected by inhibition of protein kinase C, particularly cancer tumors,inflammatory disease, myocardial reperfusion injury, and cardiacdysfunctions related to reperfusion injury. Inhibition of protein kinasec can lead to inhibition of growth of cells and can thereby produce ananti-tumor effect. Further, inhibition of protein kinase c can also leadto inhibition of the oxidative burst in neutrophils, plateletaggregation, and keratinocyte proliferation, whereby ananti-inflammatory effect is achieved. The inhibitory activities of thecompounds of the invention against platelet aggregation, neutrophilactivation, and neutrophil release demonstrate their usefulness intreating reperfusion injury, particularly myocardial reperfusion injury.

The compounds of the invention are expected to be particularly useful inthe treatment of tumors resistant to treatment with otherchemotherapeutic agents. Surprisingly, compounds of the invention areable to inhibit protein kinase C even in adriamycin or Mitoxantroneresistant cells, and thus are not cross-resistant with thesechemotherapy agents.

Compounds of the invention are able to inhibit proliferation of tumorcells at low concentrations (less than 1 μM) which should lessen thenpotential for deleterious side effects of compounds of the inventionwhen administered for treatment of tumors.

The compounds of the invention appear to have no effect on cAMPdependent protein kinase C and should consequently have no effect on themetabolic pathways associated with stimulation of protein kinase C bycAMP.

This invention is more particularly pointed out in the appended claimsand is described in its preferred embodiments in the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthetic route of Scheme I which is used for synthesisof compounds of the invention.

FIG. 2 shows the synthetic route of Scheme II which is used forsynthesis of compounds of the invention.

FIG. 3 shows the synthetic route of Scheme III which is used forsynthesis of compounds of the invention.

FIG. 4 shows a graphical representation of the median MCF-7 tumor weightof control and mice treated with compound 1b-1 vs. days post implant ofthe tumor. Tumor size for control mice is shown by solid circles; tumorweight for test mice is shown by solid squares.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel substituted anthraquinones havingFormula I ##STR3##

R₁ and R₂ are preferably independently H, C₁ -C_(1O) alkyl, aryl,arylalkyl, alkylaryl, or R₁ and R₂ taken together with the adjacentnitrogen atom N form a substituted or unsubstituted cyclic ring; morepreferably H or lower alkyl; most preferably lower alkyl. m and n arepreferably independently 1, 2, or 3; more preferably 1 or 2; mostpreferably 1. A is preferably halogen, OH, alkoxy, OCO(NR₃ R₄),S--C(NH₂)═NR₅, or when m=1, comprises an oxirane ring with the adjacentoxygen atom; more preferably halogen, OCO(NR₃ R₄), SC(NH₂)═NR₅, or whenm=1, comprises an oxirane ring with the adjacent oxygen atom; mostpreferably Cl, Br, OCO(NR₃ R₄), SC(NH₂)═NR₅, or when m=1, comprises anoxirane ring with the adjacent oxygen atom. Halogen as used hereinincludes fluorine, chlorine, bromine, and iodine. R₃, R₄, and R₅ arepreferably independently H, alkyl, or aryl; more preferably H or alkyl;most preferably H. X is preferably H, OH NR₆ R₇, Cl, Br, I, F, alkyl,aryl, alkoxy, aryloxy, COOR₈, or CONR₉ R₁₀ ; more preferably H, HO, Cl,Br, I, F, or alkoxy; most preferably H or OH. R₆, R₇, R₇, R₉, and R₁₀are independently H, lower alkyl, or aryl. As used herein, the termlower alkyl is intended to mean C₁ -C₆ alkyl.

As used herein, alkyl substituents include straight chain, branched andcyclic moieties, preferably straight chain species. While saturatedspecies are preferred, unsaturated sites may find utility in somesubstituents. Alkaryl substituents are preferably substituted benzenemoieties, with ortho, meta, and para substituents each believed to beuseful. Multiple substitutions are similarly useful. Aralkylsubstituents are preferably benzene substituted alkanes includingbenzyl, phenylethyl, phenylpropyl, etc.

The compounds of the invention are useful for treating conditionsrelated to, or affected by inhibition of protein kinase C, particularlycancer tumors, inflammatory disease, reperfusion injury, and cardiacdysfunctions related to reperfusion injury. Accordingly, another aspectof the invention provides methods and pharmaceutical compositions forinhibiting protein kinase C which comprise contacting protein kinase Cwith an inhibitory amount of a substituted anthraquinone compound of theinvention. The pharmaceutical compositions of the invention comprise asubstituted anthraquinone compound of the invention and apharmaceutically acceptable carrier of diluent.

The invention also provides methods of inhibiting an oxidative burst inneutrophils which comprise contacting a neutrophil with a protein kinaseC inhibitory concentration of a substituted anthraquinone compound ofthe invention, or contacting the neutrophil with an amount of a compoundof the invention effective to inhibit such oxidative outburst.

The invention further provides methods for treating inflammation whichcomprise administering to a mammal suffering from inflammation a proteinkinase C inhibitory concentration of a substituted anthraquinonecompound of the invention, or administering to the mammal an amount of acompound of the invention effective to inhibit inflammation.

The invention additionally provides methods for inhibiting growth ofmammalian tumor cells which comprises contacting a mammalian tumor cellwith a protein kinase C inhibitory concentration of a substitutedanthraquinone compound of the invention, or contacting the tumor cellwith an amount of a compound of the invention effective to inhibitgrowth of the tumor cell.

An additional embodiment of the invention provides methods for treatingmammalian tumors which comprise administering to a mammal having a tumora protein kinase C inhibitory concentration of a substitutedanthraquinone compound of the invention, or administering to the mammalhaving a tumor an amount of a compound of the invention effective toinhibit growth of the tumor.

Another embodiment of the invention provides methods of inhibitingmammalian keratinocyte proliferation which comprises administering to amammalian keratinocyte a protein kinase C inhibitory amount of acompound of the invention, or administering to the keratinocyte anamount of a compound of the invention effective to inhibit proliferationof the keratinocyte.

An additional embodiment of the invention provides the use of asubstituted anthraquinone compound of the invention in the preparationof a medicament for inhibiting protein kinase C. The compound of theinvention is preferably present in the medicament in a therapeuticallyeffective amount.

Surprisingly, the compounds of the invention are not cross-resistant toadriamycin (Doxorubicin HCl, Adria Laboratories, Dublin, Ohio, anantibiotic-derived chemotherapy agent) or Mitoxantrone (Novantrone©,Lederle Laboratories, Pearl River, N.Y., a synthetic anthracene dionechemotherapy agent), and remain effective in reducing growth of tumorcells resistant to these well-known chemotherapy agents. The lack ofcross-resistance with adriamycin or Mitoxantrone indicates that thecompounds of the invention will be particularly useful for treatment ofadriamycin or Mitoxantrone resistant tumors.

Cancer is a disease characterized in part by uncontrolled cell growth.Protein kinase C is directly involved in cellular growth control and isbelieved to be involved in tumor formation. Protein kinase C is themajor, if not exclusive, intracellular receptor of phorbol esters whichare very potent tumor promoters. Phorbol esters and other tumorpromoters bind to and activate protein kinase C. Since diacylglycerol(DAG) and phorbol esters interact at the sane site, DAG's have beensuggested to be the "endogenous phorbol esters" by analogy with theopiate receptor where the conservation of a high affinity receptorimplied the existence of an endogenous analogue. DAG has been shown toincrease the affinity of protein kinase C for Ca⁺² and phospholipid andthus activates protein kinase C at cellular levels of these essentialcofactors. Extracellular signals including hormones, growth factors, andneurotransmitters are known to stimulate phosphatidylinositol turnoverresulting in the generation of IP₃ and DAG. Structures of 40 distinctoncogenes of viral and cellular origin have revealed that oncogenesencode altered forms of normal cellular proteins. Several of the geneproducts appear related to growth factors or other elements involved intransmembrane signalling. These oncogene products appear to function byaltering the level of critical second messengers. Cells transformed withthe oncogenes ras, sis, erbB, abl, and src have been shown to containelevated levels of DAG which is then believed to activate protein kinaseC. Indeed studies on ras transformed cells have shown protein kinase Cactivation to concomitant with elevation of DAG.

Phorbol esters, such as phorbol myristate acetate (PMA), have complexeffects on cells including effects on membrane function, mitogenesis,differentiation, and gene expression. Synthetic diacylglycerols mimicmany of the effects of PMA in vitro and inhibitors of protein kinase Chave been shown to block PMA-induced effects on cells. Thus, proteinkinase C may mediate the actions of certain oncogenes, such as ras,which cause intracellular increases in DAG and concomitant increases inprotein kinase C. In addition, activation of protein kinase C leads tothe expression of c-myc, c-fos, c-cis, c-fms, nuclear protooncogenesimportant in cell transformation. Overexpression of protein kinase C inNIH 3T3 cells causes altered growth regulation and enhancedtumorigenicity and in rat fibroblasts leads to anchorage-independentgrowth in soft agar. In these experiments, overexpression of proteinkinase C in these cells resulted in tumor formation in animals receivingtransplanted cells.

Several studies have shown increased expression of protein kinase C incertain tumor types such as breast and lung carcinomas. Activatedprotein kinase C has also been detected in human colon carcinomasalthough increased expression on the gene level was not seen.Topoisomerases are directly modulated by protein kinase C as substratesfor the enzyme and protein kinase C inhibitors have been shown topotentiate the action of chemotherapy drugs such as cis-platinum.

New and more potent compounds which have been identified specifically asinhibitors of protein kinase C are showing early promise as therapeuticagents in inhibiting tumor growth in animal models.

Animal studies have shown that perhaps 50% or more of ischemic-relatedmyocardial damages can be attributed to polymorphonuclear leukocytes(neutrophils) which accumulate at the site of occlusion. Damage from theaccumulated neutrophils may be due to the release of proteolytic enzymesfrom the activated neutrophils or the release of reactive oxygenintermediates (ROI). Much of the "no reflow" phenomenon associated withmyocardial ischemia is attributed to myocardial capillary plugging. Theplugging of capillaries has been attributed to both aggregated plateletsand aggregated neutrophils. Although both cell types are aggregatedduring the ischemic event, the relative contribution of each tocapillary plugging has not yet been established. It is well acceptedthat the damage by neutrophils to myocardial tissue proceeds through acascade of events, one of the earliest being the bonding of activatedneutrophils to damaged vascular endothelium. However, the binding of theneutrophils is significantly enhanced by their activation and this aneven earlier event is the generation of molecules (such as cytokines,and chemotactic factors) which can function as activation stimuli. Thesemolecules probably originate from damaged and aggregated platelets, fromdamaged vascular endothelium, or from the oxidation of plasma proteinsor lipids by endothelial-derived oxidants.

Strategies for overcoming the deleterious effects of reactive oxygenintermediates have centered in the development of scavengers for themolecules. Superoxide dismutase (SOD) has been shown to be aparticularly effective scavenger of superoxide, but suffers from a veryshort half-life in the blood. Several companies have tackled thisproblem by creating versions of this enzyme with increased half-lives bytechniques such as liposome encapsulation or polyethylene glycolconjugation. Reports on the effectiveness of these new version aremixed. Catalase, a scavenger of hydrogen peroxide, and hydroxyl radicalscavengers have also been tested and found to be effective to varyingdegrees. However, none of the strategies designed to scavenge reactiveoxygen intermediates will prevent the aggregation of platelets, therelease of chemotactic molecules, the activation and adherence ofneutrophils to vascular endothelium, or the release of proteolyticenzymes from activated neutrophils.

The advantage of protein kinase C inhibitors as therapeutics forreperfusion injury is that they have been demonstrated to 1) blockplatelet aggregation and release of neutrophil activating agents such asPAF, 2) clock neutrophil activation, chemotactic migration, andadherence to activated or damaged endothelium, and 3) block neutrophilrelease of proteolytic enzymes and reactive oxygen intermediates. Thusthese agents have the capability of blocking all three of the mostsignificant mechanisms of pathogenesis associated with reperfusioninjury and should thus have a decided therapeutic advantage.

The compounds of the invention may be made according to the methods inScheme I, II and III, as shown in FIGS. 1, 2 and 3, respectively. Othersynthetic routes known in the art may also be used. As used herein, theanthraquinone ring structure has the conventional ring numbering asillustrated in the Merck Index, Tenth Edition, Merck & Co., Inc. Rahway,N.J., 1983, pp. 100-101.

When A is halo or comprises an oxirane ring with the adjacent oxygenatom, the compounds of the invention may be prepared according to themethod in Scheme I shown in FIG. 1. In Scheme I, reaction ofdiaminoanthraquinone 1 with epihalohydrin producesbis-(3-halo-2-hydroxypropylamino)-anthraquinone 2 which upon treatmentwith amine, such as diethylamine, at temperatures from room temperatureto the boiling temperature of the chosen solvent, yields the product Ia.The epoxide analog Ib was obtained by treating Ia with base such assodium hydroxide. An alternative procedure, shown in Scheme II in FIG.2, is the conversion of the bis-halohydrin intermediate 2 via basictreatment to his-epoxide 3 followed by reaction of 3 with amine to yieldIb. Examples of compounds which may be prepared according to Schemes Iand II are listed in Tables 1 and 2.

When A is a functional group other than halo or comprising an oxiranering with the adjacent oxygen, the compounds of the invention listed inTable 1 may be prepared according to Scheme III as shown in FIG. 3. Forexample, as shown in FIG. 3, compound Ia from Scheme I is converted fromthe halo compound to the hydroxy compound Ic. The hydroxy compound isthen used for the synthesis of compounds having the formula of I whereinA is alkoxy, OCO (NR₃ R₄) or SC (NH₂ ═NR₅ using the method disclosed inKocovsky et al. (1986) Tetrahedron Letters 27: 5521, the disclosures ofwhich are hereby incorporated by reference as if fully set forth herein.Other methods known in the art are also suitable for the synthesis ofcompounds of the invention.

The optically active enantiomers of the compounds of the invention canbe prepared according to Schemes 1 and 2, using optically activestarting materials such as the commercially available glycidol orepichlorohydrin.

Pharmaceutically acceptable salts of the compounds of the invention arealso useful in the methods of the invention. Pharmaceutically acceptablesalts useful in the invention include salts of hydrochloric acid,hydrobromic acid, fumaric acid, oxalic acid, malic acid, succinic acid,pamoic acid, sulfuric acid and phosphoric acid.

                                      TABLE 1                                     __________________________________________________________________________    Compound                                                                            R.sup.1                                                                           R.sup.2                                                                           X    A        m n MP (°)C.)                              __________________________________________________________________________    1a-1  Et  Et  H    Cl       1 1 106-108 (HCl)                                 1a-2  H   Pr  H    Cl       1 1 Hyg                                           1a-3  H   CH.sub.2 Ph                                                                       H    Cl       1 1 Hyg                                           1a-4  Octyl                                                                             Octyl                                                                             5-Cl Br                                                         1a-5  Et  Ph  6-Ph-O                                                                             I        2 3                                               1c-1  Et  Et  H    OH       1 1 Hyg                                           1d-1  Et  Et  H    OCH.sub.3                                                                              1 1                                               1d-2  Me  Et  7-HO O-(n-Bu) 2 2                                               1e-1  Et  Et  H    O.sub.2 CNH.sub.2                                                                      1 1                                               1e-2  Me  Me  8-Br O.sub.2 CNPr.sub.2                                                                     1 2                                               1f-1  Et  Et  H    SC(NH.sub.2)═NH.sub.2                                                              1 1                                               1f-2  n-Bu                                                                              n-Bu                                                                              F    SC(NH.sub.2)═NMe.sub.2                                                             3 3                                               1f-3  n-Bu                                                                              Me  5-COOH                                                                             C(NH.sub.2)═NPr.sub.2                                                              1 2                                               __________________________________________________________________________     (Abbreviations used in Tables 1 and 2: Me-methyl; Et-ethyl; Pr-propyl;     Ph-phenyl; Bu-butyl; Hyg-hygroscopic)

                  TABLE 2                                                         ______________________________________                                        Compound R.sup.1                                                                              R.sup.2 X      m    n   MP (°C.)                       ______________________________________                                        1b-1     Et     Et      H      1    1   98(d)                                 1b-2     H      Pr      H      1    1                                         1b-3     n-Bu   n-Bu    5-HO   2    1                                         1b-4     Pr     Me      5-Br   2    2                                         1b-5     H      H       7-COOH 3    2                                         1b-6     Me     Me      8-Me   1    1                                         1b-7     n-Bu   Me      6-NMe2 1    1                                         ______________________________________                                    

The compounds of the invention may be administered by any method thatproduces contact of the active ingredient with the agent's site ofaction in the body of a mammal including but not limited to oral,intravenous, and intraparenteral. The compounds of the invention may beadministered singly, or in combination with other compounds of theinvention, other pharmaceutical compounds, such as chemotherapycompounds, or other therapies, such as radiation treatment. Thecompounds are preferably administered with a pharmaceutically acceptablecarrier selected on the basis of the selected route of administrationand standard pharmaceutical practice.

The compounds of the invention are administered to mammals, preferablyhumans, in therapeutically effective amounts or concentrations which areeffective to inhibit protein kinase C, or to inhibit tumor cell growth,inhibit inflammation of tissue, inhibit keratinocyte proliferation,inhibit oxidative burst from neutrophils or inhibit plateletaggregation. The dosage administered in any particular instance willdepend upon factors such as the pharmacodynamic characteristics of theparticular compound of the invention, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency oftreatment, and the effect desired. It is contemplated that the dailydosage of the compounds will be in the range of from about 5 to about400 mg per kg of body weight, preferably from about 10 to about 200 mgper kg body weight, and more preferably from about 10 to about 50 mg perkg per day, and preferably administered in divided doses 2 to 4 times aday or in sustained release form.

The compounds of the invention may be administered as medicaments orallyin solid dosage forms, such as capsules, tablets, and powders, or inliquid dosage forms, such as elixirs, syrups, and suspensions. They mayalso be administered parenterally in sterile liquid dosage forms.

The compounds of the invention may be formulated for use as medicamentsinto dosage forms according to standard practices in the field ofpharmaceutical preparations. See Remington's Pharmaceutical Sciences, A.Osol, Mack Publishing Company, Easton, Pa., a standard reference text inthis field.

For example, the compounds of the invention may be mixed with powderedcarriers, such as lactose, sucrose, mannitol, starch, cellulosederivatives, magnesium stearate, and stearic acid for insertion intogelatin capsules, or for forming into tablets. Both tablets and capsulesmay be manufactured as sustained release products for continuous releaseof medication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration may contain coloring andflavoring to increase patient acceptance, in addition to apharmaceutically acceptable diluent such as water, buffer or salinesolution.

For parenteral administration, the compounds of the invention may bemixed with a suitable carrier or diluent such as water, a oil, salinesolution, aqueous dextrose (glucose), and related sugar solutions, andglycols such as propylene glycol or polyethylene glycols. Solutions forparenteral administration contain preferably a water soluble salt of thecompound of the invention. Stabilizing agents, antioxidizing agents andpreservatives may also be added. Suitable antioxidizing agents includesodium bisulfite, sodium sulfite, and ascorbic acid, citric acid and itssalts, and sodium EDTA. Suitable preservatives include benzalkoniumchloride, methyl- or propyl-paraben, and chlorbutanol.

Examples

The following are specific examples which are illustrative of thepresent invention and are not intended to limit the scope of theinvention.

EXAMPLE 1

1,4-Bis-(3Chloro-2-Hydroxypropylamino)-9, 10-Anthracenedione

Epichlorohydrin (84.5 mL, 1.08 mol) was added to a solution of 1,4-diaminoanthraquinone (Aldrich Chemical Company, Milwaukee, Wis.) (10 g,42 mmol) in glacial acetic acid (200 mL) at room temperature. Thesolution was stirred for 30 minutes at 90° C. and the solvent wasremoved under reduced pressure. The residue was purified by flash columnchromatography (silica gel, CH₂ Cl₂ :MeOH/25:1) and recrystallized fromCH₂ Cl₂ -Ether-Hexanes to give a pure blue solid of the title compound(12.46 g, 70%), mp 167°-169° C.: ¹ H NMR (CDCl₃) δ3.21 (broad, 2H, OH) ,3.5-3.6 (m, 6H, CHCH₂), 3.75 (d, J=3 Hz, 4H, CH₂), 4.19 (broad, 2H, NH),7.03 (d, J=3.2 Hz, 2H, ArH (2, 3)), 7.69 (m, 2H, ArH (6, 7)), 8.27 (m,2H, ArH (5, 8)). Anal. Calcd for C₂₀ H₂₀ O₄ N₂ Cl₂ ×0.25 H₂ O: C, 56.15;H, 4.83; N, 6.55. Found: C, 56.18; H, 4.76; N, 6.29.

EXAMPLE 2

1-(3-Diethylamino-2-Hydroxypropylamino)-4-(Chloro-2-Hydroxypropylamino)-9,10 Anthracenedione (Compound 1a-1 in Table 1)

Diethylamine (3.15 mL, 60.86 mmol) was added dropwise over 1.5 hours toa solution of 1,4-bis-(3-Chloro-2-hydroxypropylamino)-9, 10anthracenedione (12.88 g, 30.43 mmol) the compound of Example 1,compound 2 in Schemes I and II. in ethanol (200 mL) under nitrogenatmosphere. The solution was stirred for 48 hours at reflux and thesolvent was removed under reduced pressure. The residue was purified byflash column chromatography (silica gel, CH₂ Cl₂ :MeOH/25:1) andrecrystallized from CH₂ Cl₂ -ether-hexanes to give a blue solid of thetitle compound1-(3-diethylamino-2-hydroxypropylamino)-4-(chloro-2-hydroxypropylamino)-9,10 anthracenedione which is compound 1a-1 in Table I (8.1 g, 60%), mp165° C.: ¹ H NMR (CDCl₃) δ1.10 (t, J=7 Hz, 6H, CH₃) 2.63 (m, 4H, CH2),2.74 (m, 2H, CH₂ ), 3.44 (m, 2H, CH₂), 3.56 (m, 1H, CH), 3.63 (m, 1H,CH), 3.73 (m, 2H, CH₂), 4.17 (broad, 1H, NH), 4.19 (broad, 1H, NH), 7.22(s, 2H, ArH (2, 3)), 7.68 (m, 2H, ArH (6, 7)), 8.3 (m, 2H, ArH (5,8)).Anal. Calcd for C₂₄ H₃₀ O₄ N₃ Cl *1.25 H₂ O: C, 59.74; H, 6.69; N, 8.71.Found: C, 59.50; H, 6.27; N, 8.50.

EXAMPLE 3

1-(3-Diethylamino-2-Hydroxypropylamino)-4-(3-Chloro-2-Hydroxypropylamino)-9,10-Anthracenedione Hydrochloride Salt (Compound 1a-1 in Table 1)

Concentrated HCl (8 drops) was slowly added to a solution of1-(3-diethylamino-2-hydroxypropylamino)-4-(3-chloro-2-hydropropylamino)-9,10-anthracenedione (0.531 g, 2.36 mol) in acetone-dichloromethane (50%,15 mL). A color change from purple to red was observed. The solid wasobtained by filtration, mp 106°-108° C., extremely hygroscopic. ¹ H NMR(D₂ O) δ1.40 (t, J=7.3 Hz, 6H, CH₃), 2.90 (m, 4H, CH₂), 3.29 (m, 4H,CH₂), 3.38 (m, 3H, CH₂, CH), 3.73 (m, 3H, CH₂, CH), 3.92 (broad, 1H,NH), 4.15 (broad, 1H, NH), 6.23 (s, 2H, ArH (2, 3)), 7.26 (m, 2H, ArH(6, 7)), 7.32 (m, 2H, ArH (5, 8)). Anal. Calcd for C₂₄ H₃₀ ClN₃ O₄ 2HCl1.5 H₂ O: C, 51.48; H, 6.30; N, 7.50. Found: C, 51.70; H, 6.35; N, 7.41.

EXAMPLE 4

1,4-Bis-(2, 3-Epoxypropylamino)-9, 10-Anthracenedione

Sodium hydroxide (3.5 g) was added to a solution of1,4-bis-(3-chloro-2-hydroxypropylamino)-9, 10-anthracenedione (8.3 g,19.8 mmoles), the compound of Example 1, in MeOH (500 mL) at 60° C. Theresulting blue solution was stirred at room temperature for 4 hours, andthe solvent was removed under reduced pressure. The residual solid wasthen purified by flash column chromatography (silica gel, CH₂ Cl₂ :MeOH/25:1). Recrystallization from CH₂ Cl₂ : hexanes yielded the titlecompound 1,4-bis-(2, 3-epoxypropylamino)-9, 10-anthracenedione which iscompound 3 in Scheme 2 (6.2 g, 89.37%), mp 189°-190° C.: ¹ H NMR (CDCl₃)δ2.74 (m, 1H, CH₂), 2.87 (m, 1H, CH₂), 3.26 (m, 1H, CH), 3.57 (m, 1HCH₂), 3.78 (m, 1H, CH₂), 7.32 (s, 2H, Ar (2,3)), 7.71 (dd, J=3.2 Hz, 5.5Hz, 2H, Ar (6, 7)), 8.34 (dd, J=3.2 Hz, 5.5 Hz, 2H, Ar (5, 8)), 10.76(s, 2H, NH). Anal. Calcd for C₂₀ H₁₈ O₄ N₂ ×0.25 H₂ O: C, 67.66; H,5.25; N, 7.93. Found: C, 67.72; H, 5.55; N, 7.54. IR: 3400, 3070, 2910,1640, 1220, 900.

EXAMPLE 5

1-(3-Diethylamino-2-Hydroxypropylamino)-4-(2, 3-Epoxypropylamino)-9,10-Anthracenedione (Compound 1b-1 of Table 1)

Potassium hydroxide (0.3 g, 5.36 mmole) was added to a solution of1-(3-diethylamino-2-hydroxypropylamino)-4-(3-chloro-2-hydroxypropylamino)-9,10-anthracenedione hydrochloride salt (1 g, 1.78 mmole), the compound ofExample 3, in methanol. The mixture was stirred at room temperature for48 hours. Solvent was removed under reduced pressure and the residue waspurified by flash column chromatography (silica gel, chloroform with amethanol gradient up to a ratio of 10:1) to give the title product1-(3-diethylamino-2-hydroxypropylamino)-4-(2, 3-epoxypropylamino)-9,10-anthracenedione which is compound 1b-1 of Table 1 (391 mg, 52%), mp98° C.: NMR (CDCl₃) δ1.06 (t, J=7.1 Hz, 6H, CH₃), 2.62 (m, 7H, CH₂),2.87 (m, 1H, CH₂), 3.27 (m, 1H, CH₂), 3.49 (m, 2H, CH₂), 3.59 (m, 1HCH₂), 3.77 (m, 1H, CH), 3.96 (m, 1H, CH), 7.35 (s, 2H, Ar (2, 3)), 7.71(m, 2H, Ar (6, 7)), 8.35 (m, 2H, Ar (5, 8)), 10.80 (b, 1H, NH), 10.92(b, 1H, NH); Anal. Calcd for C₂₄ H₂₉ N₃ O₄ : C, 68.1; H, 6.90; N, 9.92.Found: C, 67.86; H, 6.96; N, 9.85.

EXAMPLE 6

1-(3-Diethyl-2-Hydroxypropylamino)-4-(2, 3-Dihydroxypropylamino)-9,10-Anthracenedione Hydrochloride Salt (HCl salt of compound 1C-1 ofTable 1)

Para-toluenesulfonic acid (272 mg, 1.43 mmole) was added to a solutionof 1-(3-diethylamino-2-hydroxypropylamino)-4-(2, 3-epoxypropylamino)-9,10 anthracenedione (120 mg, 0.28 mmole), the compound of Example 5, inmethanol-water (1:1, 20 mL). The mixture was stirred for 16 hours atroom temperature. The solvent was removed under reduced pressure and theresidue was treated with chloroform-water. The organic layer was driedover magnesium sulfate and, after the removal of solvent, the residuewas purified by column chromatography (silica gel, CH₂ Cl₂ with anincreasing methanol gradient to give the product as a free base.Hydrochloride gas was passed through a methanolic solution of the freebase until a color change was noted. The title compound1-(3-diethyl-2-hydroxypropylamino)-4-(2, 3-dihydroxypropylamino)-9, 10anthracenedione hydrochloride salt was then isolated by filtration andwas found to be extremely hygroscopic. NMR (CDCl₃) δ1.06 (t, J=7.1 Hz,6H, CH₃), 2.72-2.55 (m, 6 H, CH₂), 3.19 (m, 4H, CH₂), 3.31 (b, 3H, OH),3.81 (m, 2H, CH), 3.87 (b, 1H, OH), 3.92 (m, 2H, CH₂), 4.11 (b, 1H, OH), 6.65 (m, 2H, Ar (2, 3)), 7.52 (m, 2H, (6, 7)), 8.08 (m, 2H, (5, 8)),10.73 (m, 2H, NH);

EXAMPLE 7

Protein Kinase C Inhibition

The protein kinase C (PKC) assay is designed to duplicated the in vivoconditions required for protein kinase C function. Therefore, pH, saltand cofactor concentrations are similar to physiologic levels. HistoneH1 (lysine rich) is used in the assay as the phosphorylation acceptorprotein because it is readily available and serves as a good substratefor protein kinase C. The enzyme is prepared from rat brain and ispurified to apparent homogeneity as determined by a single band onsilver stained SDS-polyacrylamide. Studies on the mechanism ofregulation of protein kinase C by phospholipids, DAG and Ca⁺² have beenhampered by the physical properties of the lipid cofactors. In thescreening assay, phosphatidylserine (PS) and DAG are cosonicated to formunilamellar and multilamellar vesicles. The concentration of lipids inthe assay are suboptimal to maximize the detection potential of theassay for inhibitors. Potential inhibitor compounds are added to theassay in dimethylsulfoxide at three concentrations to give finalinhibitor concentrations of 4.3, 43 and 218 μM, respectively. The assayis started with the addition of enzyme and stopped after 10 min by theaddition of 25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serumalbumin (BSA). The radioactive histone product is retained and washed onglass fiber filters that allow the unreacted ³² P-ATP to pass through.The amount of phosphorylation is determined by the radioactivitymeasured in a scintillation counter. Controls are included in everyassay to measure background activity in the absence of enzyme, activityin the absence of lipids and the maximum enzyme activity with saturatinglevels of the activator lipids. Assay components and concentrations aregiven in Table 3.

                  TABLE 3                                                         ______________________________________                                        Assay Component  Concentration                                                ______________________________________                                        Hepes pH 7.5     20         mM                                                MgCl.sub.2       20         mM                                                CaCl.sub.2       100        μM                                             EGTA             95         μM                                             Histone H1       200        μg/ml                                          Phosphatidylserine                                                                             40         μg/ml                                          Diacylglycerol   1.8        μg/ml                                          Protein Kinase C 0.6        μg/ml                                          γ-.sup.32 P-ATP                                                                          20         μM                                             ______________________________________                                    

Results of the protein kinase C assay are shown in Table 4 in the columnlabeled protein kinase C. Results are shown as IC₅₀, which is theconcentration of test compound needed to inhibit 50% of the proteinkinase C activity as compared with levels of protein kinase C activityin controls. As shown in Table 4, all but one of the compounds testedwas able to inhibit fifty percent of protein kinase C activity atconcentrations less than 200 μM. Compound 1b-1 (base) had the lowestIC₅₀ of 110 μM, whereas compound 1a-1 (HCl salt) had the highest IC₅₀ ofthe test compounds at 230 μM.

                  TABLE 4                                                         ______________________________________                                        IC.sub.50 (μM)                                                             Exp.             PKC    PKA                                                   ______________________________________                                        1a-1 (base)      180    n.e.                                                  1a-1 (HCl)       230    n.e.                                                  1a-2 (HCl)       160    n.e.                                                  1a-3 (HCl)       110    n.e.                                                  1c-1 (HCl)       180    n.e.                                                  1b-1 (base)      110    n.e.                                                  ______________________________________                                         (In Table 4, n.e. = no effect)                                           

EXAMPLE 8

cAMP Dependent protein Kinase (PKA) Assay

Compounds found to be inhibitors of protein kinase C were also testedfor inhibitory activity against cAMP dependent protein kinase (PKA).This enzyme, like protein kinase C, plays an important role in cell-cellcommunication and is activated by a second messenger, cAMP. Secondaryscreening against PKA is useful for ascertaining the selectivity of thecompounds of the invention. The standard assay conditions are given inTable 5. The catalytic subunit of PKA (Sigma Chemical Company, St.Louis, Mo.) is mixed with buffer before addition of the inhibitor indimethylsulfoxide (DMSO). The assay is started by the addition of ³²P-ATP and the reaction is allowed to proceed for 10 min before stoppingwith 25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin(BSA). The phosphorylated protein is isolated by filtration and theradioactivity is counted in a beta scintillation counter.

                  TABLE 5                                                         ______________________________________                                        Assay Components                                                                              Concentration                                                 ______________________________________                                        Hepes pH 7.5    20          mM                                                Histone         200         μg/ml                                          Dithiothreitol  32          μg/ml                                          Protein Kinase  2.6         μg/ml                                          γ-.sup.32 -ATP                                                                          20          μM                                             ______________________________________                                    

Results of the PKA assay are shown in Table 4 (in Example 7) in thecolumn labeled PKA. As shown in Table 4, the compounds of the inventionthat were tested had no effect on PKA. The tested compounds of theinvention are selective for protein kinase c, and have no effect on cAMPdependent protein kinase. The compounds of the invention should thushave no effect on the metabolic pathways associated with stimulation ofprotein kinase by cAMP.

EXAMPLE 9

Human Tumor Growth Inhibition

MCF-7 a human breast tumor cell line, and MCF-7/ADR an adriamycinresistant line of MCF-7 cells were obtained from the National CancerInstitute, Frederick, Md. NHEK a human keratinocyte cell line wasobtained from Clonetic Corp., San Diego, Calif. 8226 a human myelomacell line, 8226/MT a Mitoxantrone resistant line of 8226 cells, and 8226ADR an adriamycin resistant line of 8226 cells were obtained from Dr.Bill Dalton, Arizona Cancer Center, Tuscon, Ariz.

Human tumor cells are trypsinized (0.05% trypsin, GIBCO), counted with ahemacytometer and seeded at a concentration of 10,000 cells/well in a 96well microtiter plate. After allowing cells to attach to the surfaceovernight, the culture medium is aspirated and replaced with 100 μl offresh medium. Test agents are diluted to determine dose response at 2×final concentration and added in quadruplicate at 100 μl/well to bringthe total volume of each well to 200 μl. The microtiter plate is thenincubated at 37° C. 5% CO₂ overnight (18-24 hrs) before ³ H-thymidine isadded at a concentration of 0.5 μCi/well in 50 μl culture medium. Theplate is incubated again for 4 hrs under the same conditions as above.Supernatant is then aspirated and 50 μl trypsin (0.05%, GIBCO) is addedto each well. Cells are checked microscopically to determine detachmentfrom surfaces, and plates are then harvested with a cell harvester (PHD,Cambridge Technology, Inc.) Filter papers corresponding to wells areplaced in scintillation vials and counted to determine the amount of ³H-thymidine incorporated by the cells. Test agent response is comparedto a positive control of cell wells with culture media only to determinethe IC₅₀. IC₅₀ is the concentration of test compound required to inhibitfifty percent of the incorporation of ³ H-thymidine into proliferatingcells not exposed to test agent. Uptake of ³ H-thymidine is a standardtest for measuring the metabolism of cells. Cells which are activelyproliferating take up ³ H-thymidine, whereas cells that are notproliferating take up ³ H-thymidine at much slower rates or not at all.Test agents that inhibit the uptake of ³ H-thymidine thus slow thegrowth of cells.

Results of these experiments are shown in Tables 6 and 7. As shown inTable 6, both compounds 1a-1 and 1b-1 were able to inhibit fifty percent of ³ H-thymidine uptake at concentrations less than 1 μM (IC₅₀=0.25 μM and 0.025 μM, respectively). The IC₅₀ of compound 1b-1especially compares well with the IC₅₀ of adriamycin.

When compounds of the invention were tested with adriamycin resistantcell line MCF-7/ADR, the IC₅₀ of the test compounds increased to amountsfrom 0.06 to 16 μM, resulting in a resistance ratio of IC₅₀ forMCF-7/ADR cells to IC₅₀ for MCF-7 cells of from 1.9 to 2.4. In contrast,the IC₅₀ for MCF-7/ADR cells cultured with adriamycin was 4 μM, with aratio of 200. The IC₅₀ for MCF-7/ADR cells cultured with Mitoxantronewas also 4 μM, but the ratio of IC₅₀ for MCF-7/ADR cells to IC₅₀ forMCF-7 cells was 800.

Similar results were obtained when the compounds were tested with celllines 8226, 8226/MT (Mitoxantrone resistant) and 8226/ADR (adriamycinresistant). Compounds 1a-1 and 1b-1 were able to inhibit ³ H-thymidineuptake at very low concentrations. The IC₅₀ for compound 1a-1 was 1 μM.The IC₅₀ for compound 1b-1 was 0.06 μM. The IC₅₀ for the other compoundstested were 10.2 and 15 μM. When compounds 1a-1 and 1b-1 were testedwith cell line 8226/MT the IC₅₀ was 0.24 and 0.008, respectively, with aratio of IC₅₀ for 8226 cells to IC₅₀ for 8226/MT cells of 0.24 and 0.13respectively. These results compared favorably with the results obtainedfrom cells cultured with adriamycin or Mitoxantrone. The ratio of IC₅₀so for 8226/MT cells to IC₅₀ for 8226 cells for cells cultured withadriamycin was 1 μM. The ratio of IC₅₀ for 8226/MT cells to IC₅₀ for8226 cells for cells cultured with Mitoxantrone was 3.5.

Compounds tested with adriamycin resistant 8226 cells also showed asimilar results. The test compound s were not cross-resistant withadriamycin as shown by the ratio of IC₅₀ for 8226/ADR cells to IC₅₀ for8226 cells ranging from 0.08 μM to 1.4 μM for test compounds comparedwith resistance ratios of 11.4 μM and 6 μM for adriamycin andmitoxantrone, respectively.

These results show that the compounds of the invention are effective ininhibiting the proliferation of tumor cells and are not cross-resistantwith adriamycin or mitoxantrone.

                  TABLE 6                                                         ______________________________________                                        Compound                                                                              MCF-7      MCF-7/ADR  Resistance Ratio                                ______________________________________                                        1a-1    0.25       0.5        2                                               1a-2    7.4        16         2.2                                             1a-3    7.4        14.1       1.9                                             1b-1    0.025      0.06       2.4                                             ADR     0.02       4          200                                             MT      0.005      4          800                                             ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Com-                  Resistance       Resistance                             pound 8226   8226/MT  Ratio    8226/ADR                                                                              Ratio                                  ______________________________________                                        1a-1  1      0.24     0.24     0.08    0.08                                   1a-2  10.2                     14.1    1.38                                   1a-3  15                       16.3    1.08                                   1b-1  0.06   0.008    0.13     0.016   0.26                                   1c-1  18                       25      1.4                                    ADR   0.05   0.05     1        0.57    11.4                                   MT    0.03   0.105    3.5      0.18    6                                      ______________________________________                                    

EXAMPLE 10

Human Keratinocyte Inhibition

Proliferating keratinocytes (NHEK cells purchased from Clonetics, Inc.San Diego, Calif.) in second passage were grown in Keratinocyte GrowthMedium (KGM) (Clonetics, Inc.) Cells were trypsinized with 0.025%trypsin (Clonetics), counted with a hemacytometer (Scientific Products),and seeded at a concentration of 2,500 cells/well in a 96 wellmicrotiter plate. After allowing cells to attach to the surfaceovernight, the culture medium is aspirated and replaced with 100 μl offresh KGM. Test agents were evaluated and IC₅₀ 's are determinedaccording to the ³ H-thymidine incorporation procedures described as inExample 9.

The results of these experiments are shown in Table 8. These resultsindicate that compounds of the invention are active against humankeratinocytes, and will be useful in treating topical inflammatoryconditions such as psoriasis and other conditions wherehyperproliferation of keratinocytes is a symptom.

                  TABLE 8                                                         ______________________________________                                        Compound      IC.sub.50 (μM)                                               ______________________________________                                        1a-1          0.16                                                            1b-1          0.04                                                            ______________________________________                                    

EXAMPLE 11

Neutrophil Superoxide Anion (O₂ -) Release Assay

Neutrophils were isolated form whole blood collected from humanvolunteers. All reagent materials were obtained from Sigma ChemicalCompany with the exception of isotonic saline (Travenol Lab., Inc.Deerfield, Ill.) and lymphocyte separation medium (Organon Teknika,Durham, N.C.).

Neutrophil Isolation

Whole blood was drawn and mixed with sodium heparin (final conc. 10units/ml) to prevent clotting. An equal volume of dextran (3.0%) inisotonic saline was added, mixed, and allowed to settle for 30 min tobind red blood cells (RBC). The supernatant was removed, underlayeredwith lymphocyte separation medium and centrifuged for 40 min at 400 xgin a centrifuge (Beckman GPR, Norcross, Ga.). The pellet was alternatelyresuspended in 0.2% and 1.6% NaCl to lyse RBCs before washing withHank's Balanced Salt Solution (HBSS). The washed pellet was resuspendedin 10 ml HBSS and placed on ice before counting on a hemacytometer.

Assay Procedure

The neutrophil cell concentration is adjusted to 2×10⁶ cells/ml withHBSS before adding 0.8 ml cells to 12×75 mm polypropylene test tubes(Fisher Scientific). Test agents are diluted to determine dose responseand added at 10×final concentration at a volume of 0.1 ml/tube induplicate. Then 10×concentrations of cytochrome C (15 mg/ml) withcatalase (3000 units/ml) either alone or containing 25 ng/ml phorbol12-myristate 13-acetate (PMA) are added at a volume of 0.1 ml/tube andincubated at 37° C. for 30 min before stopping the reaction by placingtubes on ice. Tubes are then centrifuged at 900 xg for 10 min, 0.5 mlsupernatant is removed and added to 0.5 ml H₂ O in a microcuvette.Optical density (OD) of cytochrome c is read in a spectrophotometer(Shimadzu) at 550 nm. The ΔOD of cytochrome c is obtained betweenPMA-stimulated and non-stimulated tubes, and the dose responses of thetest agents are compared to the positive controls (which contain HBSS inplace of test agents). PMA stimulates O₂ ⁻ production which reducescytochrome c. Reducing cytochrome c increases its absorbance, and thechange in OD of cytochrome c is proportional to the amount of O₂ ⁻produced by PMA stimulation. Inhibition of the O₂ ⁻ burst by testcompounds of the invention is seen as a reduction in the change inoptical density. Inhibition is expressed as IC₅₀ μM and is the amount oftest compound that will inhibit fifty percent of the PMA-stimulatedrespiratory outburst, i.e. O₂ ⁻ production.

The tested compounds were able to inhibit O₂ ⁻ production byPMA-stimulated neutrophils. As shown in Table 9, compound 1a-1 (base)had an IC₅₀ greater than 6.25 μM. Compounds 1a-1 (HCl) and 1b-1 (base)had IC₅₀ of greater than 12.5 μM and 25 μM, respectively.

                  TABLE 9                                                         ______________________________________                                        IC.sub.50 (μM)                                                             Exp.         Human Neutrophil                                                 ______________________________________                                        1a-1 (base)  >6.25                                                            1a-1 (HCl)   >12.5                                                            1b-1 (base)  >25                                                              ______________________________________                                    

EXAMPLE 12

Tumor Assays

P388 Leukemia Tumors. Mice were young adult, pathogen-free, BALA/c×DBA/2F1 (i.e., CD2F1) females purchased from Charles River Laboratories. Thetumor line was the P388 murine leukemia, ascites form, obtained from theNational Cancer Institute (NCI) Tumor Repository (Frederick, Md.). Thetumor was maintained in the serial passage in mice by periodicintraperitoneal (ip) implantation. For this experiment, 10⁶hemacytometer-counted P388 murine leukemia cells were implanted ip intomice on Day 0. The mice were randomized into one untreated control groupof 20 and 15 test groups of six mice each. All drug treatment wasdelivered ip on the basis of the average body weight of each group. Alldrugs were dissolved in sterile saline for injection, and all drugs weresoluble at all dosages prepared. Drugs, dosages, and treatment schedulesused were: mitoxantrone, a positive control drug, injected once everyfour days beginning on Day 1 for a total of three injections at 1.2,1.8, and 2.7 mg/kg/dose; Compound 1a-1 injected once every four days(Q4D×3) beginning on Day 1 for a total of three injections at 100, 150and 225 mg/kg/dose; Compound 1a-1 injected once daily for nine days(Q1D×9) beginning on Day 1 at 67, 100, and 150 mg/kg/dose; and Compound1b-1 delivered on the same treatment schedules as Compound 1a-1, but atdosages of 10.7, 16 and 24 mg/kg/dose and 3.5, 5.3, and 8 mg/kg/dose,respectively. Body weights were recorded on Days 1, 5, 9, 11, and 18.Deaths were recorded daily. Dying mice were necropsied, and the presenceor absence of ascites or splenomegaly was recorded. Drug effectivenesswas assessed on the basis of the survival time. Results were expressedas a percentage of the control survival time (Survival Time=T/C×100%;T=test animals; C=control animals). The criterion for effectiveness wasdetermined by: T/C×100≧125 percent.

                  TABLE 10                                                        ______________________________________                                        % T/C (Dose, Schedule)                                                        Compound    p388 Leukemia                                                     ______________________________________                                        1a-1        130 (150 mg/kg Q1D × 9)                                     1a-1        140 (67 mg/kg, Q1D × 9).sup.                                1b-1        140 (16 mg/kg, Q4D × 3).sup.                                ______________________________________                                    

As shown in Table 10, compounds 1a-1 and 1b-1 effectively increased thesurvival time of mice treated with the compounds at all dosages andschedules.

Melanotic Melanoma B16 Test. The animals used were B₆ C₃ F₁ mice, all ofone sex, weighing a minimum of 18 g for males and 17 g for females andall within a 4 g weight range at the start of the test. The test groupcomprised 9 or 10 mice. The tumor was implanted in each of the test miceby the subcutaneous injection of 0.5 ml of a tumor homogenate preparedby homogenizing a 1 g portion of melanotic melanoma in 10 ml of coldphysiological saline. Compound 1a-1 suspended in hydroypropylcellulosewas administered intraperitoneally at a dose of 100 mg/kg once daily fornine consecutive days staring on Day 1 relative to the day of tumorinoculation (Day 0). The control mice received injections ofhydroxyproplcellulose vehicle only. The mice were weighed and survivorswere recorded on a regular basis for sixty days. The median survivaltimes and the ratio of the median survival times for treated (T) tocontrol (C) mice were calculated. The median survival time of thenontreated rumored mice ranged from 15 to 17 days. Drug effectivenesswas assessed on the basis of survival time. Results were expressed as apercentage of the control survival time (Survival Time T/C×100%; T=testanimals; C=control animals). The criterion for effectiveness wasdetermined by T/C×100≧125 percent.

% T/C for tested compound 1a-1 was 129, indicating that compound 1a-1 iseffective for increasing survival time of the mice treated with thecompound.

MCF-7 Human Mammary Carcinoma Xenografts Mice were young adult,pathogen-free, NCrOnu (athymic nude) females purchased from TaconicFarms. MCF-7 human mammary carcinoma cells were obtained from the NCITumor Repository, expanded in culture, adapted to growth as xenograftsin nude mice, and maintained in serial passage in nude mice assubcutaneous (sc) tumors. All mice except one estrogen-free controlgroup (10 mice) received a controlled-release estradiol pellet(Innovative Research of America, Toledo, Ohio) implanted subcutaneouslyon Day 0. Tumor fragments (approximately 30 mg each) were implantedsubcutaneously by trocar on the flank opposite the estradiol implant onDay 0. The estrogen-free controls were also implanted with tumorfragments. Estrogen-implanted mice were randomized into a control groupof 30 mice and five test groups of 10 mice each. Both drugs weredissolved in sterile saline for injection, and both drugs were solubleat the dosages prepared. The five test groups received drug treatment asfollows: adriamycin, a positive control drug, was injected once everyfour days beginning on Day 1 for a total of three injections at 3.35 and5 mg/kg/dose. Compound 1b-1 was injected once every four days beginningon Day 1 for a total of five injections at 16, 25 and 40 mg/kg/dose. Alldrug treatment was administered intravenously (iv) to mice via the tailvein. Estrogen-free and estrogen-implanted controls were untreated. Bodyweights were recorded on Days 1, 5, 9, 13, 17, 21, 23 and 30. Deathswere recorded daily. Mice were palpated daily for the appearance of sctumors. Tumors measured twice weekly in two dimensions by calipers.Tumor volume was calculated using the formula for a prolate ellipsoid,and volume was converted to mass assuming unit density. Therapeuticresponse was assessed on the basis of tumor growth delay (T-C), tumorfree survival, and overall survival. Drug toxicity was assessed on thebasis of body weight loss and early (relative to the completion oftreatment) deaths.

FIG. 4 shows a graph of the median tumor weight (mg) vs. the days postimplant of the tumors. Results for the control group of mice (estrogenIMP.) are shown by solid circles; the results for compound 1b-1 areshown by solid squares. As shown in FIG. 4, tumor growth of 1b-1 treatedmice was stabilized at about day 30 at a weight significantly lower thanthe weight of the control mice. The median tumor weight for the treatedmice remained stable until about day 38 when tumor regrowth began toincrease.

We claim:
 1. A method of inhibiting protein kinase C which comprisescontacting protein kinase C with an inhibitory amount of a substitutedanthraquinone having the formula ##STR4## wherein R₁ and R₂ areindependently H, C₁ -C₁₀ alkyl, aryl, arylalkyl, or alkylaryl; n and mare independently 1, 2, or 3; A is Halogen, OH, alkoxy, OCO(NR₃ R₄),S--C(NH₂)═NR₅, or when m=1, A represents the attachment of the oxygenatom from the adjacent hydroxyl group to form an oxirane ring; R₃, R₄,and R₅ are independently H, alkyl, or aryl; X is H, OH, NR₆ R₇, Cl, Br,I, F, alkyl, aryl, alkoxy, aryloxy, COOR₈, or CONR₉ R₁₀ ; and R₆, R₇,R₈, R₉, and R₁₀ are independently H, lower alkyl, or aryl.
 2. The methodof claim 1 wherein R₁ and R₂ independently are H or lower alkyl; n and mare independently 1 or 2; A is halogen, OCO(NR₃ R₄), SC(NH₂)═NR₅, orwhen m=1 , comprises said oxirane; R₃, R₄, and R₅ are independently H oralkyl; and X is H, HO, Cl, Br, I, F, or alkoxy.
 3. The method of claim 2wherein R₁ and R₂ independently are lower alkyl; m and n are 1; A is Cl,Br, OCO(NR₃ R₄), SC(NH₂)═NR₅, or comprises said oxirane ring; R₃, R₄,and R₅ are H; and X is H or OH.
 4. A method of inhibiting an oxidativeburst in neutrophils which comprises contacting a neutrophil with asubstituted anthraquinone having the formula ##STR5## wherein R₁ and R₂are independently H, C_(1-C) ₁₀ alkyl, aryl, arylalkyl, or alkylaryl;nand m are independently 1, 2, or 3; A is Halogen, OH, alkoxy, OCO(NR₃R₄), S--C(NH₂)═NR₅, or when m=1, A represents the attachment of theoxygen atom from the adjacent hydroxyl group to form an oxirane ring;R₃, R₄, and R₅ are independently H, alkyl, or aryl; X is H, OH, NR₆ R₇,Cl, Br, I, F, alkyl, aryl, alkoxy, aryloxy, COOR₈, or CONR₉ R₁₀ ; andR₆, R₇, R₈, R₉, and R₁₀ are independently H, lower alkyl, or aryl in anamount effective to inhibit said oxidative burst.
 5. The method of claim4 whereinR₁ and R₂ independently are H or lower alkyl; n and m areindependently 1 or 2; A is halogen, OCO(NR₃ R₄), SC(NH₂)═NR₅, or whenm=1, comprises said oxirane ring; R₃, R₄, and R₅ are independently H oralkyl; and X is H, HO, Cl, Br, I, F, or alkoxy.
 6. The method of claim 5whereinR₁ and R₂ independently are lower alkyl; m and n are 1; A is Cl,Br, OCO(NR₃ R₄), SC(NH₂)═NR₅, or comprises said oxirane ring; R ₃, R₄,and R₅ are H; and X is H or OH.
 7. The method of claim 4 wherein saidamount is a protein kinase C inhibitory amount.
 8. The method of claim 5wherein said amount is a protein kinase C inhibitory amount.
 9. Themethod of claim 6 wherein said amount is a protein kinase C inhibitoryamount.
 10. A method for treating inflammation which comprisesadministering to a mammal suffering from inflammation a substitutedanthraquinone having the formula ##STR6## wherein R₁ and R₂ areindependently H, C₁ -C₁₀ alkyl, aryl, arylalkyl, or alkylaryl;n and mare independently 1, 2, or 3; A is Halogen, OH, alkoxy, OCO(NR³ R⁴),S--C(NH₂)═NR₅, or when m=1, A represents the attachment of the oxygenatom from the adjacent hydroxyl group to form an oxirane ring; R₃, R₄,and R₅ are independently H, alkyl, or aryl; X is H, OH, NR₆ R₇, Cl, Br,I, F, alkyl, aryl, alkoxy, aryloxy, COOR₈, or CONR₁₀ ; and R₆, R₇, R₈,R₉, and R₁₀ are independently H, lower alkyl, or arylin an amounteffective to inhibit inflammation.
 11. The method of claim 10 whereinR₁and R₂ independently are H or lower alkyl; n and m are independently 1or 2; A is halogen, OCO(NR₃ R₄), SC(NH₂)═NR₅, or when m=1, comprisessaid oxirane ring; R.sub. 3, R₄, and R₅ are independently H or alkyl;and X is H, HO, Cl, Br, I, F, or alkoxy.
 12. The method of claim 11whereinR₁ and R₂ independently are lower alkyl; m and n are 1; A is Cl,Br, OCO(NR₃ R₄), SC(NH₂)═NR₅, or comprises said oxirane ring; R₃, R₄,and R₅ are H; and X is H or OH.
 13. The method of claim 10 wherein saidamount is a protein kinase C inhibitory amount.
 14. The method of claim11 wherein said amount is a protein kinase C inhibitory amount.
 15. Themethod of claim 12 wherein said amount is a protein kinase C inhibitoryamount.
 16. A method of inhibiting keratinocyte proliferation comprisingadministering to a keratinocyte a compound having the formula ##STR7##wherein R₁ and R₂ are independently H, C₁ -C₁₀ alkyl, aryl, arylalkyl,alkylaryl;n and m are independently 1, 2, or 3; A is Halogen, OH,alkoxy, OCO(NR₃ R₄), S--C(NH₂)═NR₅, or when m=1, A represents theattachment of the oxygen atom from the adjacent hydroxyl group to forman oxirane ring; R₃, R₄, and R₅ are independently H, alkyl, or aryl; Xis H, OH, NR₆ R₇, Cl, Br, I, F, alkyl, aryl, alkoxy, aryloxy, COOR₈, orCONR₉ R₁₀ ; and R₆, R₇, R₈, R₉, and R₁₀ are independently H, loweralkyl, or arylin an amount effective to inhibit proliferation of saidkeratinocyte.
 17. The method of claim 16 whereinR₁ and R₂ independentlyare H or lower alkyl; n and m are independently 1 or 2; A is halogen,OCO(NR₃ R₄), SC(NH₂)═NR₅, or when m=1, comprises said oxirane ring; R₃,R₄, and R₅ are independently H or alkyl; and X is H, HO, Cl, Br, I, F,or alkoxy.
 18. The method of claim 17 whereinR₁ and R₂ independently arelower alkyl; m and n are 1; A is Cl, Br, OCO(NR₃ R₄), SC(NH₂)═NR₅, orcomprises said oxirane ring; R₃, R₄, and R₅ are H; and X is H or OH. 19.The method of claim 11 wherein said amount is a protein kinase Cinhibitory amount.
 20. The method of claim 17 wherein said amount is aprotein kinase C inhibitory amount.
 21. The method of claim 18 whereinsaid amount is a protein kinase C inhibitory amount.